NOVEL NUTRITIONAL STRATEGIES FOR ANIMAL HEALTH

There is currently a great interest in developing alternative feeding strategies for animal health maintenance and disease prevention, in order to reduce the negative impact of removing antibiotic growth promoters (AGP) within the EU (EC Regulation 1831/2003), while minimizing the therapeutic use of antibiotics. The main theme of this research concerns the management of some health problems that frequently occur during the weaning of piglets, focusing in particular on the use of feed additives and immunogenic products derived from engineered plants. Three different trials were designed to study novel strategies for the control of two important diseases, caused by infection with Escherichia coli strains, typically occurring during the weaning phase of the piglet, specifically E. coli diarrhoea and Oedema Disease (OD). E. coli diarrhoea is a multifactorial disease responsible for heavy economic losses in pig production. The study of alternative strategies for the prevention of E. coli diarrhoea is limited by its variable incidence in the field and by the difficulty to reproduce the disease after experimental infection. For these reasons, the aim of the first study was to set up experimental conditions to simulate the outbreak of diarrhoea through a controlled E. coli challenge. Thirty-five healthy piglets, weaned at 33±2 days, from a selected farm, were divided into three experimental groups: control group (CG) including 5 piglets, infected group 1 (IG1) including 10 piglets and infected group 2 (IG2) including 20 piglets. One day after arrival, the piglets of IG1 and IG2 were orally inoculated with 3.7*10^8 CFU of E. coli O149 strain, while CG received 5 mL of sterile physiological saline. The animals were fasted for 3 hours before and after challenge and 30 mL of 10% NaHCO3 solution were individually administered 15 minutes before challenge. The IG and CG groups were fed with a diet containing a high level of protein (CP: 28%) for 3 days after infection. The health status, faecal score (0:normal; 1:soft; 2:liquid; 3:watery) and faecal colour (3:brown; 2:green; 1:yellow) were individually recorded daily for 20 days after challenge for IG1 and CG and for 2 days after challenge for IG2. Polymerase chain reaction, serotyping and biochemical identification were established for the evaluation of E. coli strains from faecal samples. The effects of the challenge were analyzed by multivariate repeated measures. Diarrhoea was observed in 96.67% (58.6% severe; 41.4% mild) of all infected piglets and occurred on average 1.3 days after challenge. The CG group presented one piglet with a transient mild diarrhoea. The E. coli challenge significantly affected the consistency and the colour of faeces (P<0.001). A significant correlation (r=-0.89; P<0.001) between faecal colour and consistency was found. 70% (14/20) of infected piglets with severe diarrhoea shed E. coli O149 in their faeces. The O149 challenger strain was detected in 17 out of 30 (56.7%) infected piglets two days after experimental infection, and 15 out of 17 isolated O149 E. coli strains (88%) were also haemolytic. Zootechnical parameters did not show significant differences. These findings support the view that, being post-weaning diarrhoea multifactorial, other factors have to be combined with the oral challenge to reproduce the disease under experimental conditions. We concluded that the experimental protocol described in this study can be used in the evaluation of nutritional strategies to prevent or control E. coli diarrhoea in weaned piglets. The two subsequent experimental works are part of a wider project, in which transgenic tobacco plants have been engineered for the seed-specific expression of antigens against verotoxin-producing E. coli (VTEC) strains responsible for OD. Novel strategies are required to control VTEC infections, considering that at present no vaccines are available and the treatment relies upon the use of antibiotics, which may contribute to the increase of antimicrobial resistance. In this context, plant-vaccines have considerable potential and represent a promising strategy for mucosal vaccination in terms of low costs, safety, storage, transportation and for the production of specific antibodies in the mucosa, where the major pathogens gain access to the body. In the present studies two different lines of tobacco seeds were previously transformed via agroinfection for the expression of two antigenic proteins of VTEC strains: the F18 fimbria, responsible for the adherence of the bacteria on small intestinal enterocytes, and the B subunits of verotoxin 2e (VT2e), responsible for binding the toxin to specific receptors on the cell surface. One of the most important issues related to the oral delivery of plant-based vaccines is the potential for antigen degradation in the gastrointestinal tract. For this reason, a preliminary trial was designed to evaluate the effect of swine gastric fluid, derived from weaned piglets, on the VT2e-B antigenic protein expressed in whole and milled tobacco seeds. Samples of transgenic tobacco seeds, both milled and whole, were incubated with porcine gastric fluid, at 38° C in a Dubnoff Shaker for 1, 2 and 3 hours. After gastric fluid removal, by centrifugation and washing with PBS, the samples were homogenized in the presence of a protein extraction buffer. Western blot was performed on representative samples of extracted proteins, quantified by the Bradford method, using rabbit polyclonal serum. The Vt2e-B specific signals were observed on SDS-page in all samples derived from transgenic tobacco seeds. Nevertheless, from 0 h to 3 h, a progressive reduction of intensity of the Vt2e-B specific signal was observed. No significant differences were detected on the reduction of signal intensity between samples derived from whole and milled tobacco seeds. Based on the results obtained, we could conclude that the residual amount of transgenic proteins after digestion of both milled and whole seeds appears sufficient for their use in immunization trials on piglets. Within the same project, the last trial was designed to evaluate the use of transgenic tobacco seeds expressing antigenic proteins of VTEC strains as edible vaccine in piglets. A total of 43 weaned piglets (20±2 days) were randomized into 4 groups. Three immunized groups (T1, T2, T3) orally received a bolus of tobacco seeds (TS) mixed with chocolate on days 0, 1, 2, 14 of the trial. In particular, the T1 group received 10 grams of TS-F18+ and 10 grams of TS-VT2e-B+, the T2 group received 10 grams of TS-VT2e-B+ and the T3 group received 25 grams of TS-VT2e-B+. Control Group (CG) received 20 grams of wild type TS. The amount of transgenic protein was estimated about 0.6 mg/gram of whole TS. In this immunization phase faecal and blood samples were collected weekly to evaluate IgA and IgG amounts by ELISA assays. On day 22, the piglets were orally challenged with 1*10^10 CFU of O138 Escherichia coli strain, using the experimental protocol described in the first trial. Faecal score, body temperature and clinical signs related to OD (palpebral oedema, epiphora, neurological and respiratory symptoms, vitality) were determined, daily for 15 days after challenge, for each piglet through specific point scales. Zootechnical performances and haematocrit percentages (HT) were evaluated during the experimental period. T1, receiving both the antigens, showed a higher level of IgA in faeces than other groups on day 21 (22,000±13,000 ng/ml vs control group: 7,200±3,000 ng/ml). No differences were observed among groups in relation to the total IgG titre in faeces and total IgA and IgG levels in serum. For each clinical sign, the average total score (the sum of the average daily score from day 1 to day 9 post-challenge) was significantly higher in the control group compared to orally immunized groups (P<0.05) and the latter showed a faster recovery than CG. In the same period (day 1 to 9 post-challenge), T1 showed a significantly higher consistency of faeces compared to T1 and T2 (P<0.05). No differences were observed in body temperature and HT. After challenge (day 21 to 25), the average daily feed intake (P<0.05) and the average daily gain were higher in T1 and T2 than CG. For all measured parameters, no differences were observed between T2 and T3, suggesting that no dose-response effect was shown for the Vt2e-B antigen in our experimental conditions. In conclusion, oral administration of recombinant tobacco seeds expressing antigenic proteins against VTEC strains can induce an increase of mucosal antibodies and a protective effect against the challenger strain in piglets. This represents a promising non-invasive method of vaccinating swine via their feed.